BIOCHEMICAL STUDIES OF ACQUIRED CHEMOTHERAPY RESISTANCE

本申请的总体目的是确定临床上 对伊立替康产生耐药性的相关机制- 局部晚期非小细胞肺癌患者的基础化疗 肺癌（NSCLC）。 伊立替康是喜树碱衍生物， 在NSCLC中具有显著活性。 目前当地的管理 晚期NSCLC包括诊断时的手术分期， 化疗治疗，然后手术切除 肿瘤，从而提供了一个独特的机会，以获得足够的 术前化疗前后的病理资料 以进行拟议的实验室研究。喜树碱 将拓扑异构酶（topo）I：DNA共价复合物稳定在 单链蛋白质连接DNA断裂（PLDB）。 PLDB可以 随后转化为dsDNA断裂， 致命的 细胞对喜树碱的耐药性可能与步骤有关 在PLDB形成之前发生的，例如 拓扑I，或由PLDB形成后发生的步骤产生， 例如增强修复DNA损伤的能力。 为了 表征对喜树碱的抗性如何在体内发展， 在三个周期的预处理之前和之后获得的NSCLC肿瘤组织 由伊立替康和顺铂组成的手术化疗将 被分析。 在由肿瘤和肿瘤细胞制备的单细胞悬浮液中， 喜树碱体外孵育，dsDNA损伤和PLDB 将分析形成。 药物敏感性比较将 确定有限暴露于非治愈性化疗是否 诱导抗性以及抗性是否在 成立PLDB。 浓度、活性、细胞内 分布，拓扑异构酶I对伊立替康的亲和力将是 以确定这些特定的生物化学物质中 改变有助于体内对伊立替康的抗性。 额外 研究将根据DNA分析是否 病变表明在治疗过程中获得的阻力 在PLDB形成之前。 完成后 第一项临床试验，第二项研究将使用 伊立替康加紫杉醇的组合。 生化研究 分析耐药的临床相关机制， 将重复使用喜树碱。 表型比较 在肿瘤中观察到的变化将提供对 伊立替康的耐药机制， 与不同种类的化疗剂组合。 在 此外，由于多药耐药（MDR）可能与 紫杉醇的活性，我们将评估这些肿瘤的 术前暴露前后MDR的表达 化疗 这些药物外排相关的过度表达 蛋白质将通过免疫细胞化学和功能 用99 Tc-Sestamidi体内扫描。 的表达是否 术前化疗后NSCLC的MDR增加 含有紫杉醇，然后选择肿瘤内的克隆 表达MDR可能与最终治疗失败有关。 这将支持更多的试验，其中调节剂是 包括在根除这些克隆的初始治疗中。 这些 生物化学方法应该能更好地理解 非小细胞肺癌化疗获得性耐药的机制 并带来治疗这种疾病的新策略。

The overall aim of this application is to identify the clinically relevant mechanisms of resistance which develop to irinotecan- based chemotherapy in patients with locally advanced non small cell lung cancer (NSCLC). Irinotecan is a camptothecin derivative with significant activity in NSCLC. Current management of locally advanced NSCLC incorporates surgical staging at diagnosis, treatment with chemotherapy, and then surgical resection of the tumor, thus providing a unique opportunity to obtain sufficient pathologic material before and after pre-operative chemotherapy in order to conduct the laboratory studies proposed. Camptothecins stabilize the topoisomerase (topo) I: DNA covalent complex in a single strand protein linked DNA break (PLDB). PLDBs can subsequently be converted to dsDNA breaks which are potentially lethal. Cellular resistance to camptothecin may be related to steps occurring before the formation of the PLDB, such as reduction in topo I, or result from steps occurring after formation of the PLDB, such as an enhanced capacity to repair DNA damage. In order to characterize how resistance to camptothecin develops in vivo, NSCLC tumor tissue obtained before and after three cycles of pre- operative chemotherapy consisting of irinotecan and cisplatin will be analyzed. In single cell suspensions prepared from tumor and incubated in camptothecin in vitro, dsDNA damage and PLDB formation will be analyzed. Comparison of drug sensitivity will determine whether limited exposure to non-curative chemotherapy induces resistance and whether resistance develops before or after formation of the PLDB. The concentration, activity, intracellular distribution, and topoisomerase I affinity for irinotecan will be characterized to determine which of these specific biochemical alterations contribute to resistance to irinotecanin vivo. Additional studies will be directed according to whether analysis of DNA lesions suggest that resistance acquired during treatment occurs before of after formation of the PLDB. Following completion of this first clinical trial, a second study will be performed utilizing the combination of irinotecan plus paclitaxel. Biochemical studies to analyze clinically relevant mechanisms of resistance to camptothecins will be repeated. Comparison of the phenotype changes observed in the tumor will provide insight into the mechanisms of resistance which are invoked toirinotecan when it is combined with different classes of chemotherapeutic agents. In addition, since multidrug resistance (MDR) may be relevant to the activity of paclitaxel, we will evaluate these tumors for the expression of MDR before and after exposure to pre-operative chemotherapy. The overexpression of these drug efflux associated proteins will be measured by immunocytochemistry and functionally by in-vivo scanning with 99Tc-sestamidi. If the expression of MDR is increased in NSCLC following pre-operative chemotherapy containing paclitaxel, then selection of clones within the tumor which express MDR may be relevant to ultimate treatment failure. This would support additional trials in which modulators are included in the initial treatment to eradicate these clones. These biochemical approaches should result in a better understanding of the mechanisms of acquired resistance to chemotherapy in NSCLC and lead to new strategies to treat this disease.